Epstein-Barr virus (EBV) infects human B cells and induces their immortalization. The molecular basis for EBV immortalization is largely unknown. Recently, it was observed that when EBV immortalized cells are cultured without serum at critically low densities, proliferation ceases and the cells die. However, addition of cell-free supernatants from exponentially growing EBV-immortalized cell lines can rescue these cells from death and promote their proliferation. Recently, we have identified IL-6 as being one of the autocrine growth factors for EBV-immortalized B cells (Tosato, G. et al, J. Virol., 1990) However, only approximately 25% of autocrine growth factor activity is attributable to IL-6 while the remaining autocrine growth factor activity resides in a low molecular weight component. We sought to identify this low molecular weight activity. The purification was accomplished, sequentially, by gel filtration chromatography, exchange chromatography, adsorption and gel permeation chromatography, reverse phase HPLC and hydrophilic-interaction HPLC. The material purified in this manner was analyzed by mass spectrometry. Two major components were identified as lactic acid (LA) and oxalic acid. Synthetic LA stimulated growth in EBV-immortalized B cells at 1 to 10 mM, a concentration of LA achieved in the culture supernatant of EBV- immortalized cell lines. This molecule alone was found to account for all the low molecular weight autocrine growth factor activity in supernatants of EBV-immortalized B cells. Thus, LA is an autocrine stimulatory molecule that induces the proliferation of EBV-immortalized B cells.